Optical arrangement and scan microscope

ABSTRACT

An optical arrangement for spatially separating an illumination light beam and a detection light beam includes an acousto-optical component that splits the detection light beam by birefringence. A compensation element is provided that compensates, in a single passage of the detection light beam, for the splitting of the detection light beam.

The invention relates to an optical arrangement for spatially separatingan illumination light beam and a detection light beam, with anacousto-optical component.

Furthermore, the invention relates to a scanning microscope having alight source that generates an illumination light beam and having adetector that picks up a detection light beam coming from a specimen,and also having an acousto-optical component for spatially separatingthe illumination light beam and the detection light beam.

BACKGROUND

In scanning microscopy, a specimen is illuminated with a light beam inorder to observe the detection light emitted by the specimen asreflection or fluorescent light. The focus of an illumination light beamis moved in a specimen plane by means of a controllable beam deflector,normally by tilting two mirrors, whereby the deflection axes are usuallyat right angles with respect to each other so that one mirror deflectsin the x direction while the other deflects in the y direction. Themirrors are tilted, for example, by means of galvanometer positioningelements. The output of the detection light coming from the object ismeasured as a function of the position of the scanning beam. Normally,the positioning elements are fitted with sensors in order to ascertainthe actual position of the mirror. The illuminating light is coupled inby means of a beam splitter. The fluorescent or reflection light comingfrom the object passes through the beam splitter and subsequentlyreaches the detectors.

Especially in the case of confocal scanning microscopy, an object isscanned in three dimensions with the focus of a light beam.

A confocal scanning microscope normally comprises a light source, afocusing lens system with which the light from the source is focusedonto a pinhole diaphragm—the so-called excitation diaphragm—a beamsplitter, a deflector for controlling the beam, a microscope lenssystem, a detection diaphragm and the detectors to pick up the detectionor fluorescent light. The illuminating light is coupled in by means of abeam splitter. The fluorescent or reflection light coming from theobject returns via the deflector to the beam splitter, passes throughthe latter and is subsequently focused on the detection diaphragm behindwhich the detectors are located. This detector arrangement is called adescanning arrangement. Any detection light that does not come directlyfrom the region of focus takes a different light path and does not passthrough the detection diaphragm, so that point information is obtained,which produces a three-dimensional image as a result of sequentialscanning of the object with the focus of the illumination light beam. Inmost cases, a three-dimensional image is obtained by means of image dataacquisition one layer at a time.

Leica's German Preliminary Published Application DE 199 06 757 A1discloses an optical arrangement in the optical path of a light sourcesuitable for fluorescence excitation, preferably in the optical path ofa confocal laser scanning microscope, having at least one spectrallyselective element for coupling the excitation light of at least onelight source into the microscope and for blocking out the excitationlight that is scattered and reflected off the object, or else theexcitation wavelength coming from the object via the detection opticalpath. For purposes of attaining a variable configuration with a simpledesign, the arrangement is characterized in that excitation light havingdifferent wavelengths can be blocked out by the spectrally selectiveelement. As an alternative, such an optical arrangement is characterizedin that the spectrally selective element can be adjusted with respect tothe excitation wavelength that is to be blocked out. Moreover, the citedpublication states that the spectrally selective element can beconfigured as an AOTF (acousto-optical tunable filter) or as an AOD(acousto-optical deflector). The above-mentioned preliminary publishedapplication states that the spectrally selective element can cause aspatially spectral spreading out which can be compensated for, forinstance, with three additional optical components.

German Preliminary Published Application DE 198 59 314 A1 discloses anarrangement of a light-diffraction element for separating excitation andemission light in the optical path of a microscope, preferably in aconfocal microscope, and especially in a laser scanning microscope,whereby the light-diffraction element is traversed by the excitationlight as well as by the emission light and influences at least onewavelength of the excitation by means of diffraction, whereas otherwavelengths emitted by the specimen pass through the element withoutbeing affected, as a result of which they are spatially separated fromthe excitation light. This arrangement comprises an AOTF.

German Preliminary Published Application DE 199 44 355 A1 discloses anoptical arrangement in the optical path of a laser scanning microscopewith at least one spectrally selective element that can be adjusted tothe wavelength of the excitation light of a light source, whereby saidelement couples excitation light of the light source into themicroscope, blocks the excitation light that is scattered and reflectedoff an object out of the detection optical path and does not block outthe detection light coming from the object. In order to simplify thedesign of the known arrangement as well as to expand the detectionvariants that are possible so far, this optical arrangement ischaracterized in that, downstream from the element, there is anotheroptical component and, after it has been traversed, the dispersiveand/or birefringent properties of the detection light are combined insuch a way that they can be detected and, in a preferred embodiment,this is done coaxially.

In comparison to scanning microscopes where the illuminating light andthe detection light are separated by means of a beam splitter, thescanning microscopes mentioned above entail the advantage of spectralflexibility since the acousto-optical component can be adjusted to anydesired optical wavelength for the illumination or detection light bymeans of actuation with sound waves of different frequencies. Inaddition, the spectral separation of these microscopes is many timesbetter than that of scanning microscopes with beam splitters.

A drawback of optical arrangements having an acousto-optical componentfor separating the illuminating light and the detection light as well asof scanning microscopes having an acousto-optical component forseparating the illuminating light and the detection light lies in thefact that the acousto-optical component is birefringent, which leads toa detrimental splitting of the detection light beam. Moreover, theacousto-optical component usually has a prism effect, which causes aspectral splitting of the detection light beam. The known arrangementsdo not compensate for this effect adequately and they entail high lossesof detection light output. Especially arrangements that call for threeadditional optical components for the compensation are expensive andtheir adjustment is complex.

SUMMARY OF THE INVENTION

Therefore, an object of the invention is to provide an opticalarrangement that allows a separation of an illumination light beam andof a detection light beam in a way that incurs few losses and largelywithout causing detrimental splitting phenomena.

The present invention provides a optical arrangement for spatiallyseparting an illumination light beam and a detection light beam. Theoptical arrangement includes an acousto-optical component capable ofsplitting the detection light beam by birefringence. A compensationelement is also provided. The compensation element is configured tocompensate, in a single passage of the detection light beam, for thesplitting of the detection light beam. It is likewise an object of theinvention to provide a scanning microscope with which detrimentalsplitting phenomena can be largely compensated for while entailing fewlosses.

The present invention also provides a scanning microscope. The scanningmicroscope includes a light source, a detector, an acousto-opticalcomponent, and a compensation element. The light source is configured togenerate an illumination light beam. The detector is configured todetect a detection light beam coming from a specimen. Theacousto-optical component is configured to spatially separate theillumination light beam and the detection light beam, theacousto-optical component being capable of splitting the detection lightbeam by birefringence. The compensation element is configured tocompensate, in a single passage of the detection light beam, for thesplitting of the detection light beam.

The invention has the advantage that it can utilize the universality andflexibility of acousto-optical components for separating an illuminationlight beam and a detection light beam largely without any restrictions.

In a preferred embodiment of the scanning microscope or of the opticalarrangement, the acousto-optical component is configured as an AOTF(acousto-optical tunable filter). Likewise feasible embodiments arethose with an AOD (acousto-optical deflector) or an AOM (acousto-opticalmodulator). The acousto-optical element is traversed by a sound wavewhich, as a function of its frequency, only interacts with onewavelength. Light having other wavelengths remains unaffected by thesound wave. The sound wave is preferably created by an electricallyactuated piezo sound generator that is actuated by a high-frequencywave. The value of the high frequency is selected in such a way thatonly the fractions of the desired wavelength of the illumination lightbeam reach the specimen. The other fractions of the illumination lightbeam that are not affected by the acoustic excitation are diverted intoa beam trap. The output of the beam coupled out of the illuminationlight beam can be selected by varying the amplitude of the sound wave.If the detection light beam contains florescent light—which is by natureshifted in the wavelength with respect to the illumination lightbeam—then the detection light beam passes through the acousto-opticalcomponent without being affected by the sound wave.

In a preferred embodiment, the compensation element at least largelycompensates for a splitting of the detection light beam caused bydispersion brought about by the acousto-optical component. In aparticularly simple version, the boundary surfaces of the compensationelement are arranged in such a way that the spectrally split detectionlight beam that diverges in a fan-shaped manner runs at least parallelafter having passed through the compensation element. The distancebetween the acousto-optical component and the compensation element inthis version is selected so as to be as small as possible in order toavoid excessive spatial splitting of the detection light beam betweenthe acousto-optical component and the compensation element. Spatialsplitting phenomena in the order of magnitude of half the diameter ofthe beam are acceptable.

In a preferred embodiment, the compensation element and theacousto-optical component have the same exterior shape. The compensationelement and the acousto-optical component are oriented so as to beturned by 180° with respect to each other in terms of the direction ofpropagation of the detection light beam that strikes the acousto-opticalcomponent. The compensation element thus oriented is typically laterallyoffset with respect to the axis defined by the direction of propagationof the detection light beam that strikes the acousto-optical component,so that the detection light beam strikes the compensation element.Preferably, the compensation element is made of the same material as theacousto-optical component and it has the same crystal structure. Thesplitting of the detection light beam into partial beams havingdifferent polarization caused by the birefringent properties of theacousto-optical component is eliminated.

In a preferred embodiment, the acousto-optical component is not actuatedonly with the first high frequency corresponding to the desiredwavelength of the illumination light beam since said acousto-opticalcomponent only completely blocks out the fractions of the wavelength ofthe illumination light beam having one polarization direction. Thefractions having the other polarization direction are almost completelyblocked out by actuating the acousto-optical component with another highfrequency that differs from the first high frequency.

In another embodiment, the compensation element is anotheracousto-optical component. It is particularly advantageous to alsoactuate the compensation unit with a high frequency in order to blockout fractions having the wavelength of the illumination light beam thathave remained in the detection light beam.

In another embodiment, a provision is made for a temperaturestabilization of the acousto-optical component or of the compensationelement. In another embodiment variant, in order to avoid the drawbackscaused by temperature fluctuations or fluctuations in the wavelength ofthe illumination light beam, the high frequency should be controlled orregulated as a function of the temperature. In realizing this objective,another variant provides for the wavelength of the illumination lightbeam to be controlled or regulated as a function of the temperature.

In a preferred embodiment, a device to effectuate a displacement of thebeam is arranged downstream from the acousto-optical component and fromthe compensation element. As a result, the axis of the detection lightbeam entering the optical arrangement is coaxial or at least parallel tothe axis of the detection light beam leaving the optical arrangement.This simplifies the adjustability of the optical arrangement or of thescanning microscope; furthermore, the arrangement in a scanningmicroscope can be more easily replaced by a conventional beam splitterin this configuration.

In a preferred embodiment, the acousto-optical component is cemented tothe compensation element. In another embodiment, an intermediate elementis provided that is cemented to the acousto-optical component and to thecompensation element. The cemented embodiments entail the advantage thatit is possible to largely avoid losses of detection light output causedby reflections on the boundary surfaces of the acousto-optical componentand of the compensation element that face each other. Preferably, thereis an entrance window for an illumination light beam on the intermediateelement.

BRIEF DESCRIPTION OF THE DRAWINGS

The subject matter of the invention is depicted schematically in thefigures, whereby elements having the same function are provided with thesame reference numerals. The following is shown:

FIG. 1 a scanning microscope;

FIG. 2 an optical arrangement;

FIG. 3 another optical arrangement;

FIG. 4 another optical arrangement; and

FIG. 5 another optical arrangement.

DETAILED DESCRIPTION

FIG. 1 shows a scanning microscope according to the invention, which isconfigured as a confocal microscope, having two lasers 1, 3 whoseemission light beams 5, 7, which have different wavelengths, arecombined with the dichroitic beam recombiner 9 so as to form oneillumination light beam 11. The scanning microscope has anacousto-optical component 13 that is configured as an AOTF 15. Theillumination light beam 11 is reflected by a reflecting mirror 12towards the acousto-optical component 13. From the acousto-opticalcomponent 13, the illumination light beam 11 reaches a beam deflectionmeans 17 that contains a gimbal-mounted scanning mirror 19 and guidesthe illumination light beam 11 through the scanning lens system 21,through the tubular lens system 23 and through the objective lens 25over or through the specimen 27. The detection light beam 29 coming fromthe specimen passes in the reverse direction through the scanning lenssystem 21, through the tubular lens system 23 and through the objectivelens 25, thus going via the scanning mirror 19 and reaching theacousto-optical component 13 which guides the detection light beam 29 toa compensation element 31 that is configured as an additionalacousto-optical component 33. After passing through the compensationelement 31, the detection light beam 29 strikes a pair of mirrorsconsisting of a first mirror 35 and a second mirror 37. This pair ofmirrors serves to bring the detection light beam 29 to the desired beamaxis, namely, the beam axis that is defined by the detection light beam29 as it exits from the beam deflection means 17. The pair of mirrorsguides the detection light beam 29 to the detector 39, which isconfigured as a multi-band detector. In the drawing, the illuminationlight beam 11 is depicted as a solid line and the detection light beam29 as a broken line. For the sake of completeness, the illuminationpinhole 41 commonly found in a confocal scanning microscope as well asthe detection pinhole 43 have also been drawn in schematically. On theother hand, for the sake of clarity, a few optical elements that serveto guide and shape the light beams have been omitted. These elements aresufficiently familiar to the person skilled in the art. Theacousto-optical component 13, which serves to select the fractions ofthe illumination light beam of the chosen wavelengths, is configured asan AOTF 15, which is traversed by a sound wave. This sound wave isgenerated by an electrically actuated piezo sound generator 45. Theactuation is done by means of a high-frequency source 47 that generatesan electromagnetic high-frequency wave having several adjustable HFfrequencies. The electromagnetic high-frequency wave is transmitted viaa coaxial cable 48. The HF frequencies are chosen in such a way thatonly the fractions of the desired wavelengths of the illumination lightbeam 11 reach the beam deflection means 17. The other fractions of theillumination light beam 11 that are not affected by the acousticexcitation are diverted into a beam trap. The output of the light havingthe desired wavelengths of the illumination light beam 11 can beselected by varying the amplitude of the acoustic wave. In this context,the crystal section and the orientation of the acousto-optical component13 are selected in such a way that, with the same coupling-in direction,different wavelengths are deflected in the same direction. The otheracousto-optical component 33 is likewise configured as an AOTF and it isactuated by another high-frequency source 51 having anotherelectromagnetic high-frequency wave. The high-frequency of the otherelectromagnetic high-frequency wave is selected in such a way that thefractions of the detection light beam 29 that have the wavelength of theillumination light beam 11 are blocked out. A computer 53 is providedfor purposes of selecting the HF frequencies. In accordance with theuser instructions, this computer 53 controls the high-frequency source47 and the other high-frequency source 51. With the computer mouse 55,the user establishes the appropriate settings. A slider 59, 61, 63 thatserves to set the amplitude is shown on the monitor 57 for each selectedHF frequency.

FIG. 2 shows an optical arrangement according to the invention. Thelinearly polarized illumination light beam 11, which has a sagittaldirection of polarization, is reflected by a reflecting mirror 12towards an acousto-optical component 13 that is configured as an AOTF15. This acousto-optical component 13, which serves to select thefractions of the illumination light beam of the chosen wavelengths, isconfigured as an AOTF 15 that is traversed by an acoustic wave. Theacoustic wave is generated by an electrically actuated piezo soundgenerator 45. The actuation is done by means of a high-frequency source47 that generates an electromagnetic high-frequency wave having severaladjustable HF frequencies. The electromagnetic high-frequency wave istransmitted via a coaxial cable 48. The illumination light beam 11 exitsthe acousto-optical component 13 at a direction of polarization turnedby 90°, in other words, at a tangential direction of polarization. Adetection light beam 29 enters the acousto-optical component coaxiallyto the exiting illumination light beam 11. The detection light beamcontains fractions with a sagittal as well as a tangential direction ofpolarization. The detection light beam passes through the AOTF 15, aprocess in which fractions having the wavelength of the illuminationlight beam are largely blocked out. This blocking out, however, is onlycomplete for the fractions having a tangential direction ofpolarization. After passing through the AOTF 15, the detection lightbeam 29 strikes a compensation element 31 that is configured as anotheracousto-optical component 33. This other acousto-optical component 33 islikewise configured as an AOTF and is actuated by another high-frequencysource 51 having another electromagnetic high-frequency wave. The HFfrequency of the other electromagnetic high-frequency wave is selectedin such a way that the fractions of the detection light beam 29 havingthe wavelength of the illumination light beam 11 and a sagittaldirection of polarization are blocked out. The sound wave is generatedin the compensation element 31 likewise with an electrically actuatedpiezo sound generator 65. The compensation element 31 and theacousto-optical component 13 have the same exterior shape and the samecrystal structure. The compensation element 31 and the acousto-opticalcomponent 13 are oriented so as to be turned by 180° with respect toeach other relative to the direction of propagation of the detectionlight beam 29 that strikes the acousto-optical component. As a rule, thecompensation element thus oriented is offset laterally with respect tothe axis defined by the direction of propagation of the detection lightbeam that strikes the acousto-optical component, so that the detectionlight beam strikes the compensation element.

FIG. 3 shows another optical arrangement essentially corresponding tothe arrangement described in FIG. 2. In addition, a pair of mirrors 67is provided as a device to effectuate a displacement of the beam. Afterpassing through the compensation element 31, the detection light beam 29strikes the pair of mirrors 67 consisting of a first mirror 35 and asecond mirror 37. The pair of mirrors serves to bring the detectionlight beam 29 to the desired axis 69.

FIG. 4 shows another optical arrangement. With this arrangement, thereis an intermediate element 71 that is cemented to the acousto-opticalcomponent 13 and to the compensation element 31. In this arrangement, nodetrimental reflections occur on the boundary surfaces of thecompensation element 31 and of the acousto-optical component 13 thatface each other. The index of refraction of the intermediate element 71and that of the cement are adapted to the indices of refraction of thecompensation element 31 and of the acousto-optical component 13. A glassblock 73 having the highest possible index of refraction is positioneddownstream from the compensation element 31, as a device to effectuate adisplacement of the beam; on the one hand, said glass block 73 refractsthe detection light beam 29 to the desired axis and, on the other hand,it compensates for a spectral splitting caused by the acousto-opticalcomponent 13 or by the compensation element 31.

FIG. 5 shows another optical arrangement. The acousto-optical componentin this arrangement is configured in such a way that the illuminationlight beam entering the acousto-optical component and the detectionlight beam exiting the component each have an entrance and exit windowof their own. This optical arrangement entails the advantage that, eventhough the detection light beam undergoes a spectral spreading out, thespread-out detection light beam runs virtually parallel between theacousto-optical component and the compensation element, which improvesthe compensation by the compensation element.

The invention has been described with reference to a special embodiment.However, it goes without saying that changes and modifications can beundertaken without departing from the scope of protection of the claimspresented below.

1. An optical arrangement for spatially separating an illumination lightbeam and a detection light beam, comprising: an acousto-opticalcomponent capable of splitting the detection light beam bybirefringence; a compensation element configured to compensate, in asingle passage of the detection light beam, for the splitting of thedetection light beam; and an intermediate element disposed between theacousto-optical component and the compensation element, the intermediateelement being configured to direct the illumination light beam to theacousto-optical component; wherein the compensation element and theacousto-optical component are oriented so as to be turned by 180° withrespect to each other relative to a direction of propagation of thedetection light beam striking the acousto-optical component.
 2. Theoptical arrangement as recited in claim 1 wherein the acousto-opticalcomponent includes at least one of an acousto-optical tunable filter, anacousto-optical deflector, and an acousto-optical modulator.
 3. Theoptical arrangement as recited in claim 1 wherein: the acousto-opticalcomponent is capable of splitting the detection light beam bydispersion; and the compensation element is configured to compensate forthe splitting of the detection light beam due to dispersion.
 4. Theoptical arrangement as recited in claim 1 wherein the compensationelement and the acousto-optical component have a same exterior shape. 5.The optical arrangement as recited in claim 1 wherein the compensationelement includes another acousto-optical component.
 6. The opticalarrangement as recited in claim 1 further comprising a beam-displacementdevice disposed downstream from the acousto-optical component and thecompensation element.
 7. The optical arrangement as recited in claim 1wherein the acousto-optical component and the compensation element arecemented to each other.
 8. The optical arrangement as recited in claim 1wherein the intermediate element is cemented to the acousto-opticalcomponent and the compensation element.
 9. A scanning microscopecomprising: a light source configured to generate an illumination lightbeam; a detector configured to detect a detection light beam coming froma specimen; an acousto-optical component configured to spatiallyseparate the illumination light beam and the detection light beam, theacousto-optical component being capable of splitting the detection lightbeam by birefringence; a compensation element configured to compensate,in a single passage of the detection light beam, for the splitting ofthe detection light beam; and an intermediate element disposed betweenthe acousto-optical component and the compensation element, theintermediate element being configured to direct the illumination lightbeam to the acousto-optical component; wherein the compensation elementand the acousto-optical component are oriented so as to be turned by180° with respect to each other relative to a direction of propagationof the detection light beam.
 10. The scanning microscope as recited inclaim 9 wherein the acousto-optical component includes at least one ofan acousto-optical tunable filter, an acousto-optical deflector, and anacousto-optical modulator.
 11. The scanning microscope as recited inclaim 9 wherein: the acousto-optical component is capable of splittingthe detection light beam by dispersion; and the compensation element isconfigured to compensate for the splitting of the detection light beamdue to dispersion.
 12. The scanning microscope as recited in claim 9wherein the compensation element and the acousto-optical component havea same exterior shape.
 13. The scanning microscope as recited in claim 9wherein the compensation element includes another acousto-opticalcomponent.
 14. The scanning microscope as recited in claim 9 furthercomprising a beam-displacement device disposed downstream from theacousto-optical component and the compensation element.
 15. The scanningmicroscope as recited in claim 9 wherein the acousto-optical componentand the compensation element are cemented to each other.
 16. Thescanning microscope as recited in claim 9 wherein the intermediateelement is cemented to the acousto-optical component and thecompensation element.
 17. The optical arrangement as recited in claim 1wherein the intermediate element includes a reflecting mirror.
 18. Thescanning microscope as recited in claim 9 wherein the intermediateelement includes a reflecting mirror.
 19. The optical arrangement asrecited in claim 1 wherein an index of refraction of the intermediateelement is adapted to respective indices of refraction of theacousto-optical component and the compensation element.
 20. The scanningmicroscope as recited in claim 9 wherein an index of refraction of theintermediate element is adapted to respective indices of refraction ofthe acousto-optical component and the compensation element.